Bottom Line:
Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported.Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22).Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

ABSTRACTCitrus canker, caused by the bacterial pathogen Xanthomonas citri ssp. citri (Xcc), has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world. Since identification of resistant plants is one of the most effective methods of disease management, the ability to screen for resistant seedlings plays a key role in the production of a long-term solution to canker. Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported. Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22). In addition to limiting disease symptoms, ROS production was also correlated with the expression of basal defense-related genes such as the pattern recognition receptors LRR8 and FLS2, the leucine-rich repeat receptor-like protein RLP12, and the defense-related gene PR1, indicating an important role for pathogen-associated molecular pattern-triggered immunity (PTI) in determining resistance to citrus canker. Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

fig1: Xcc-flg22 induced varying levels of ROS production in Citrus seedlings. Flg22-triggered ROS production was measured using luminol as a chemiluminescence probe in the following Citrus seedlings: C. maxima, C. paradisi, C. arantium, C. sinensis, Citrus × Poncirus trifoliata, and C. reticulata. Each reaction was monitored for 40 min with the ROS production peak from within the time course shown in CPS. Error bars represent the standard deviation of two independent experiments. The level of ROS was also measured for non-Xcc-flg22-induced samples as a negative control, which is a combination of five seedlings for each species used in the assay. Gray boxes represent one standard deviation of the mean while the black horizontal lines represent the mean.

Mentions:
A peptide corresponding to the flg22 of Xcc was ordered from GenScript (Piscataway, NJ, USA) with >90% purity using the amino sequence QRLSSGLRINSAKDDAAGLAIS. The peptide was resuspended in sterile water and used to measure induction of ROS. Six leaf disks from three newly expanded young leaves were punched out with a 4-mm-diameter cork borer. Leaf discs were floated abaxial side up in an individual well of a black 96-well plate with 200 µl of water per well at room temperature overnight and covered with plastic bags. The next day the water was removed, and ROS production was triggered with 200 nM flg22 applied together with 20 μM luminol and 1 μg per 100 μL of horseradish peroxidase. Luminescence was measured by the LUMIstar microplate luminometer (BMG Labtech, Cary, NC, USA). Each plate was measured over a period of 40 min. A total of 32 one-year-old seedlings from each variety were used. The assay was repeated twice. Controls lacking flg22 were included for each species with the control lane displayed in Figure 1 consisting of a combination of five data points from each of the species tested (30 total data points). Data were analyzed using the Optima microplate reader software (BMG Labtech).

fig1: Xcc-flg22 induced varying levels of ROS production in Citrus seedlings. Flg22-triggered ROS production was measured using luminol as a chemiluminescence probe in the following Citrus seedlings: C. maxima, C. paradisi, C. arantium, C. sinensis, Citrus × Poncirus trifoliata, and C. reticulata. Each reaction was monitored for 40 min with the ROS production peak from within the time course shown in CPS. Error bars represent the standard deviation of two independent experiments. The level of ROS was also measured for non-Xcc-flg22-induced samples as a negative control, which is a combination of five seedlings for each species used in the assay. Gray boxes represent one standard deviation of the mean while the black horizontal lines represent the mean.

Mentions:
A peptide corresponding to the flg22 of Xcc was ordered from GenScript (Piscataway, NJ, USA) with >90% purity using the amino sequence QRLSSGLRINSAKDDAAGLAIS. The peptide was resuspended in sterile water and used to measure induction of ROS. Six leaf disks from three newly expanded young leaves were punched out with a 4-mm-diameter cork borer. Leaf discs were floated abaxial side up in an individual well of a black 96-well plate with 200 µl of water per well at room temperature overnight and covered with plastic bags. The next day the water was removed, and ROS production was triggered with 200 nM flg22 applied together with 20 μM luminol and 1 μg per 100 μL of horseradish peroxidase. Luminescence was measured by the LUMIstar microplate luminometer (BMG Labtech, Cary, NC, USA). Each plate was measured over a period of 40 min. A total of 32 one-year-old seedlings from each variety were used. The assay was repeated twice. Controls lacking flg22 were included for each species with the control lane displayed in Figure 1 consisting of a combination of five data points from each of the species tested (30 total data points). Data were analyzed using the Optima microplate reader software (BMG Labtech).

Bottom Line:
Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported.Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22).Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.

ABSTRACTCitrus canker, caused by the bacterial pathogen Xanthomonas citri ssp. citri (Xcc), has been attributed to millions of dollars in loss or damage to commercial citrus crops in subtropical production areas of the world. Since identification of resistant plants is one of the most effective methods of disease management, the ability to screen for resistant seedlings plays a key role in the production of a long-term solution to canker. Here, an inverse correlation between reactive oxygen species (ROS) production by the plant and the ability of Xcc to grow and form lesions on infected plants is reported. Based on this information, a novel screening method that can rapidly identify citrus seedlings that are less susceptible to early infection by Xcc was devised by measuring ROS accumulation triggered by a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22) from X. citri ssp. citri (Xcc-flg22). In addition to limiting disease symptoms, ROS production was also correlated with the expression of basal defense-related genes such as the pattern recognition receptors LRR8 and FLS2, the leucine-rich repeat receptor-like protein RLP12, and the defense-related gene PR1, indicating an important role for pathogen-associated molecular pattern-triggered immunity (PTI) in determining resistance to citrus canker. Moreover, the differential expression patterns observed amongst the citrus seedlings demonstrated the existence of genetic variations in the PTI response among citrus species/varieties.